Isoprenoids are isoprene polymers that find use in pharmaceuticals, neutraceuticals, flavors, fragrances, perfume, and rubber products. Isoprene (2-methyl-1,3-butadiene) is a volatile hydrocarbon that is insoluble in water and soluble in alcohols. A commercially viable amount of isoprene can be obtained by direct isolation from petroleum C5 cracking fractions or obtained by dehydration of C5 isoalkanes or isoalkenes (Weissermel and Arpe, Industrial Organic Chemistry, 4th ed., Wiley-VCH, pp. 117-122, 2003), and the C5 skeleton can also be synthesized from smaller subunits.
Biosynthesis of isoprene occurs by two distinct metabolic pathways (Julsing et al. Appl Microbiol Biotechnol, 75:1377-1384, 2007). In eukaryotic organisms and protobionts, isoprene is synthesized through the mevalonate (MVA) pathway, whereas, in some bacteria and higher plants, isoprene is synthesized through the methylerythritol phosphate (MEP) pathway. Isoprene emission from plants is dependent on light and temperature with increases linked to leaf development. Isoprene synthase, an isoprene-producing enzyme, has been identified in Aspen trees (Silver and Fall, Plant Physiol, 97:1588-1591, 1991; and Silver and Fall, J Biol Chem, 270:13010-13016, 1995) and is believed to be responsible for the in vivo production of isoprene from whole leaves. Bacterial production of isoprene has also been reported (Kuzma et al. Curr Microbiol, 30:97-103, 1995; Wilkins, Chemosphere, 32:1427-1434, 1996), and varies in amount with the phase of bacterial growth and the nutrient content of the culture medium (U.S. Pat. No. 5,849,970; Wagner et al. J Bacteriol, 181:4700-4703, 1999).
However, the levels of isoprene obtainable through bacterial systems of the prior art are insufficient for commercial uses. Thus, there is a need for an efficient, large scale, bacterial isoprene production process to provide feedstock for the manufacture of isoprenoids.
Meanwhile, among C1 compounds, methane gas is produced at low costs from natural gas and synthetic gas which is a mixed gas of carbon monooxide, carbon dioxide and hydrogen obtained by incinerating waste such as biomass, municipal waste, and so on. Natural gas is attracting attention as a next-generation energy source, because it abundantly exists in fossil resources and generates a relatively small amount of CO2. Thus, transition from conventional petroleum to natural gas is in progress.
A methylotroph is a general name for a C1 compound assimilating microorganism that uses a carbon compound not having a C—C bond in the molecule, e.g., methane, methanol, methylamine, dimethylamine, trimethylamine or the like as a sole carbon source or energy source. Any microorganisms called methanotroph, methane-oxidizing bacteria, methanol assimilating bacteria, methanol assimilating yeast, methanol assimilating microorganism, belong to methylotrophs. Central metabolism of a methylotroph is a reaction of converting formaldehyde into an organic matter having a C—C bond after converting methanol to formaldehyde.
As methods of producing isoprene using a methanotroph, several methods that comprise externally introducing isoprene synthase were reported. However, when the foreign gene isoprene synthase is introduced into a methanotroph, there are disadvantages in that the methanotroph does not have a sufficient ability to produce isoprene, thereby isoprene production is low (US 2015-0225743; KR2015-0100666; WO2014-138419; WO2014-089436).